Plasminogen activators are a unique class of enzymes that convert plasminogen to its active enzymatic form, plasmin. Plasmin is a serine protease that degrades the fibrin networks of blood clots. Several plasminogen activators are currently being used as in vivo fibrinolytic agents in the treatment of acute myocardial infarction. One of these plasminogen activators, tissue plasminogen activator (t-PA), has a significantly enhanced ability to activate plasminogen in the presence of fibrin. Thus, when used as an in vivo fibrolytic agent, t-PA is directed to fibrin clots.
Human t-PA is a multi-domain serine protease secreted by vascular endothelial cells. Five distinct structural domains make up the 527 amino acids of the mature t-PA molecule. The molecule is synthesized as a one-chain polypeptide that can be converted to the two-chain form by a plasmin-mediated cleavage at Arginine 275-Isoleucine 276. The DNA and amino acid structure of human t-PA was described by Pennica et al., 1983, Nature 301:214. The numbering system employed by Pennica et al. is used herein. Gene mapping studies of genomic t-PA have shown that the gene encoding t-PA is comprised of twelve exons split by introns (Ny et al., 1984, Proc. Natl. Acad. Sci. USA 81:5355). These introns correspond, in part, with the domain junctions at the amino acid level.
The amino-terminal portion of the molecule contains a disulfide-linked loop referred to as the Finger domain (F). This domain is highly homologous to the Finger domain of fibronectin that provides this molecule with fibrin-binding properties. The second domain, called the Growth Factor domain (E), is highly homologous with epidermal growth factor. Similar growth factor domains occur in serine proteases such as urokinase, protein C and clotting factors IX and X. The third and fourth domains are highly disulfide-linked structures referred to as Kringles (K1 and K2). Similar Kringle structures are present in plasma proteins such as plasminogen and prothrombin. There is conflicting evidence as to whether both Kringle domains or just the second Kringle domain are involved in the fibrin-mediated activation of plasminogen. The fifth domain, located at the carboxy-terminus, is the Serine Protease domain (SP). The Serine Protease domain is homologous to similar domains in urokinase, plasma serine clotting proteases and trypsin.
There has been significant growth in the acceptance of thrombolytic therapy for the early restoration of blood flow to ischemic myocardium. Because of a short plasma half-life, recombinant tissue plasminogen activator must be administered by intravenous infusion to insure thrombolytic efficacy. Recent reviews of coronary thrombolysis suggest that the ideal properties for a new thrombolytic agent are that it be fibrin specific, have a longer plasma half-life allowing for a single injection administration, produce more rapid reperfusion, prevent reocclusion and be safer (i.e. lower bleeding risk, non-immunogenic), than streptokinase, t-PA or urokinase (Collen, D., Klin. Wochenschr. 1988; 66 (Suppl. 12):15-23; Bang et al., 1989, Annu. Rev. Pharmacol. Toxicol. 29:322-341; Minno et al., 1989, Pharmacol. Res. 21(2):153-161; Mueller et al., 1989, Med. Clin. North Am. 73(2):387-407).
Since the production of recombinant t-PA, investigators have produced modified recombinant forms of t-PA and urokinase in an attempt to learn more about these serine proteases (Krause, J., 1988, Fibrinolysis 2:133-142; Pannekoek et al., 1988, Fibrinolysis 2:123-133; Haigwood et al., 1989, Protein Engineering 2:611-620; Higgins and Bennett, 1990, Annu. Rev. Pharmacol. Toxicol. 30:91). A major emphasis in the design of derivative forms has been to increase fibrin specificity, increase circulating plasma half-life, and decrease bleeding liability compared with endogenous natural enzymes.
One derivative of native t-PA was described by Burck et al., 1990, Journal of Biological Chemistry 265(9):5176. This derivative, known as mt-PA6, was constructed by site-specific mutagenesis of the cDNA encoding human t-PA. The DNA encoding amino acids 4-175 was deleted so that upon expression the resultant t-PA derivative comprised the signal peptide and propeptide domains, the first three amino acids of mature t-PA, as well as the Kringle 2 and Serine Protease domains. mt-PA6 was found to possess greater fibrinolytic activity and a greater ability to activate thrombus bound plasminogen than native t-PA (Jackson et al., 1990, Circulation 82: 930-940).
t-PA derived from melanoma cells and produced by recombinant DNA methods from CHO cells, comprises four consensus Asparagine-linked (N-linked) glycosylation sites at amino acids 117, 184, 218 and 448. In native t-PA amino acid 184 is glycosylated only part of the time while amino acid 218 is not glycosylated (Higgins and Bennett, 1990). Thus, two glycosylation forms of native t-PA have been isolated. One form is glycosylated at amino acid 117 in the Kringle 1 domain and amino acid 448 in the Serine Protease domain. The second form is glycosylated at amino acids 117, 448 and 184.
The oligosaccharides at amino acids 184 and 448 are complex oligosaccharides while amino acid 117 is occupied by a high-mannose oligosaccharide (Spellman et al., 1989, J. of Biol. Chem. 264(24):14100). The structures of these oligosaccharides in native t-PA derived from CHO cells was described by Spellman et al., 1989. N-linked glycosylation sites are tripeptide sequences that are specifically recognized and glycosylated. Examples of tripeptide consensus sequences include asparagine-X-threonine and asparagine-X-serine, wherein X is any amino acid but proline.
When produced by eukaryotic cell culture, mt-PA6 is also found in two glycosylated forms. These two forms account for the doublet of 40 and 42 kD bands seen when the purified enzyme is analyzed by gel electrophoresis. Primary mt-PA6 is glycosylated only at amino acid 448. Variant mt-PA6 is glycosylated at amino acids 448 and 184. mt-PA6 lacks the K1 domain and, therefore, the glycosylation site at amino acid 117. No glycosylation is found at amino acid 218 in Kringle 2. Variant mt-PA6 comprises 15-25% of the mt-PA6 molecules secreted from a Syrian hamster cell line (Burck et al., supra).
Studies on the effect of altering glycosylation patterns of t-PA derivatives (wherein the Finger and Growth Factor domains had been deleted) indicate that derivatives which are partially or totally nonglycosylated have a higher fibrinolytic versus fibrinogenolytic ratio than their fully glycosylated counterparts (Hansen et al., 1988, Journal of Biological Chemistry 263(30): 15713-19). These results teach away from the method of the present invention. Diglycosylated t-PA derivatives lacking the Finger, EGF and Kringle 1 domains, especially Variant mt-PA6, provide advantages over their partially and nonglycosylated counterparts in the treatment of thromboembolic disorders. Variant mt-PA6 displays the unexpected and advantageous properties of causing less systemic conversion of plasminogen to plasmin, is markedly less prone to metabolism to its two-chain form, and has a higher fibrinolytic versus fibrinogenolytic ratio than Primary mt-PA6. Surprisingly, Variant mt-PA6 also provides a greater maintenance of coronary blood flow.
For purposes of the present invention, as disclosed and claimed herein, the following terms are defined below.
ApR--the ampicillin-resistant phenotype or gene conferring same.
ElA--an immediate-early gene product of adenovirus which can activate a poly-GT element to express enhancer activity and can also activate the BK virus enhancer.
ep--a DNA segment comprising the SV40 early promoter of the T-antigen gene, the T-antigen binding sites, and the SV40 origin of replication.
GBMT transcription control unit--a modified transcription control unit that comprises the P2 enhancer element of BK virus spaced closely to the upstream regulatory element of the major late promoter of adenovirus (MLTF), the adenovirus-2 major late promoter and a poly-GT element positioned to stimulate said promoter and a DNA sequence encoding the spliced tripartite leader of adenovirus-2. The GMBT transcription control unit is best exemplified by the approximately 900 base pair HindIII cassette found in plasmid pGTC which is found in E. coli K-12 AG1/pGTC (NRRL B-18593).
GT--enhancer system--any poly-GT element linked to a promoter, such as MLP, in which the poly-GT element does not itself possess enhancer activity but is activated as an enhancer by an immediate-early gene product of a large DNA virus, such as the ElA gene product or by any similarly activating viral gene product.
HmR--the hygromycin-resistant phenotype or gene conferring same.
IVS--DNA encoding an intron, also called an intervening sequence.
Large DNA virus--a virus that infects eukaryotic cells and has a genome greater than .about.10 kb in size, i.e., any of the pox viruses, adenoviruses, and herpes viruses.
MLP--the major late promoter of adenovirus, that is also referred to herein as the adenovirus late promoter, adenovirus-type-2 late promoter, or Ad2 late promoter.
MLTF binding site--the site in adenovirus DNA where the major late transcription factor (MLTF) binds; the MLTF is required for MLP activity.
N-linked glycosylation--the attachment of oligosaccharides to a protein through an N-glycosidic bond with the asparagine residue in an Asn-X-Ser/Thr sequence.
NeoR--the neomycin resistance-conferring gene, which can also be used to confer G418 resistance in eukaryotic host cells.
ori--a plasmid origin of replication.
pA--a DNA sequence encoding a polyadenylation signal.
Poly-GT element--a DNA sequence of (GT).sub.n -(CA).sub.n, which is illustrated herein by a sequence where n is 21, but which can also refer to sequences of varying lengths where n is greater or less than 21, and may refer to chemically synthesized (GT).sub.n -(CA).sub.n sequences or human genomic DNA fragments containing a (GT).sub.n -(CA).sub.n tract.
Reocclusion--complete cessation of blood flow after successful thrombolysis caused by reformation of thrombus and/or vasoconstriction.
Reperfusion--restoration of blood flow caused by successful thrombolysis.